The Hedgehog (Hh) family of secreted signaling proteins governs cell growth and patterning in numerous developmental processes in both vertebrates and invertebrates. Misregulation of Hh signaling activity has been implicated in many human disorders including cancer. The long-term goal of my laboratory is to understand how Hh controls cell growth and patterning, and how the graded Hh signals are transduced to elicit different developmental outcomes. The Hh signal reception system consists of two transmembrane proteins: a twelve-transmembrane protein Patched (Ptc) as the Hh receptor and a seven-transmembrane protein Smoothened (Smo) as the obligatory signal transducer. We have discovered that protein kinase A (PKA) and casein kinase 1 (CK1) positively regulate the Hh pathway by phosphorylating Smo, leading to its cell surface accumulation and conformational change to an active state. In addition, we found that Hh-induced phosphorylation promotes Smo interaction with the intracellular signaling complexes. We have also found that mammalian Smo undergoes a conformational change in response to Shh, which is regulated by a distinct set of kinases. These and other findings unveil interesting parallels between Drosophila and vertebrate Hh signaling mechanisms and provide new tools and hypotheses to dissect the Hh signaling mechanism in both systems. In this proposal, we will investigate the function and regulation of multiple Smo phosphorylation events in Hh signaling (Aim1), determine how phosphorylation regulates Smo cell surface expression (Aim 2), determine how Smo activates the intracellular signaling complex (Aim 3), and investigate the function and mechanism of Smo phosphorylation in Shh pathway activation (Aim 4). The proposed study should provide deeper understanding of the Hh signal transduction mechanism and shed new light into how graded Hh signals are translated into different developmental outcomes in both Drosophila and vertebrate systems. As abnormal elevation of Smo activity and Hh signaling contributes to many human cancers and Smo is a primary therapeutic target for drug development, our study may provide new avenues for improving diagnosis and therapeutics of Hh pathway related cancers.